Pro-inflammatory cytokines

Cytokines are bioactive proteins with low molecular weights (8,000 to 30,000 daltons) that act as mediators and modulators of the immunological response. Almost all nucleated cells are

capable of synthesizing cytokines and, in turn, of responding to them. They modulate the function and activity of cells around them to coordinate and control the inflammatory response. The cytokines act in networks, they can interact with each other to mediate additive, antagonistic or synergistic effects. The cytokine production is tightly regulated. The relative concentrations of cytokines are often associated to the physiological effects of cytokines.

Some cytokines initiate and amplify the inflammatory response, others sustain or attenuate it and some of them cause it to resolve. Based on their main biological activities, cytokines can be divided into pro-inflammatory (e.g. IL-6, TNFα or IL-1β) or anti-inflammatory (e.g. IL-10 or transforming growth factor (TGF)β) cytokines.

IL-6, TNFα and IL-17 are three pro-inflammatory cytokines contributing to the chronic inflammatory state of many autoimmune and inflammatory disorders. They are therefore attractive therapeutic targets. Inhibitors of IL-6, TNFα or IL-17 pathways are now available on the drug market. For this reason, this study focuses particularly on these three cytokines.

1.3.1 Interleukin-6

IL-6 is a four-helix protein of 184 amino acids with pleiotropic activities. IL-6 is synthetized and secreted by monocytes, macrophages, T cells, fibroblasts and endothelial cells. IL-6 binds the IL-6 receptor (IL-6R), which is not signaling competent. Indeed, IL-6 signaling is initiated upon association of the IL-6/IL-6R complex with a second receptor named glycoprotein (gp) 130 that thereupon dimerizes (Figure 2). Dimerization of gp130 leads to activation of the tyrosine kinase janus kinases (JAKs), which stimulate several intracellular signaling pathways including signal transducer and activator of transcription (STAT) 1 and STAT3 pathways and mitogen-activated protein kinase (MAPK) and phosphatidylinositiol-4,5-biphosphate 3-kinase (PI3K) pathways (Calabrese and Rose-John, 2014).

However, IL-6R is only expressed on few cell types including some leukocytes, hepatocytes, some epithelial cells (e.g. biliary epithelial cells) and non-epithelial cells (e.g. hepatic stellate cells) (Schmidt-Arras and Rose-John, 2016). However, because a soluble form of IL-6R

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smooth-muscle cells) (Calabrese and Rose-John, 2014). This increases the spectrum of IL-6 target cells. This signaling mode is called IL-6 trans-signaling and the IL-6 signaling through the membrane-bound IL-6R is the IL-6 classic signaling (Figure 2). Exploration of these two IL-6 signaling suggests that the IL-6 classic signaling is important for regenerative and protective functions of IL-6 whereas IL-6 trans-signaling is associated with the IL-6 pro-inflammatory activities (Scheller et al., 2011).

In human blood, a soluble form of gp130 (sgp130) is also present and acts as a natural inhibitor of IL-6 trans-signaling without affecting the IL-6 classic signaling. High levels of sgp130 are founded in the circulation of healthy individuals (250-400 ng/mL) compared to the levels of sIL-6R (40-60 ng/mL) and IL-6 that is even lower (1-5 pg/mL). Therefore, sIL-6R and sgp130 constitute an effective blood buffer for IL-6 (Calabrese and Rose-John, 2014).

Interestingly, individuals carrying a polymorphism on the IL-6R gene increasing the sIL-6R levels in blood are protected from several autoimmune diseases and cardiovascular diseases (Ferreira et al., 2013; Rafiq et al., 2007; Scheller and Rose-John, 2012). The level of sIL-6R is consequently crucial in the IL-6 overall activity since the increase of the sIL-6R level increases the capacity of the IL-6 buffer in blood.

FIGURE 2: Signaling of IL-6 via the membrane-bound and soluble IL-6 receptor

(a) In classic IL-6 signaling, IL-6 binds the membrane-bound IL-6R on hepatocytes and some leukocytes. The IL-6−IL-6R complex then associates with the signal transducing protein gp130, which is ubiquitously expressed. This association induces dimerization and IL-6 signal transduction.

(b) In trans-signaling, IL-6 binds the soluble IL-6 receptor (sIL-6R) generated by translation from an alternatively spliced mRNA or the cleavage of membrane-bound IL-6R by the metalloprotease ADAM10 or ADAM17. The IL-6−sIL-6R complex associates with the membrane-bound gp130 on cells that do not express the membrane bound IL-6R and induces dimerization and IL-6 signaling pathway. Soluble gp130 (sgp130), present in the circulation in healthy conditions, blocks the IL-6 trans-signaling by binding the IL-6−sIL-6R complex without affecting the classical signaling.

Adapted from Liu et al., 2016 - DOI: 10.1097/BOR.0000000000000255, with permission from RightsLink / Wolters Kluwer Health, Inc; License Number: 4390181057524

IL-6 is a pleiotropic cytokine with multiple functions in the body. It contributes to host defense against pathogens but plays also an important role in various autoimmune and inflammatory diseases such as rheumatoid arthritis (Tanaka et al., 2012). By acting on a wide variety of cells, IL-6 exerts multiple biological activities. IL-6 is the major inducer of the hepatic acute-phase proteins (Heinrich et al., 1990; Schmidt-Arras and Rose-John, 2016). IL-6 promotes also T cell differentiation toward Th17 cells and immunoglobulin synthesis in activated B cells (Tanaka et al., 2012). IL-6 is also involved in the chronicity of the inflammatory response by inducing mononuclear cell recruitment to the site of inflammation (Gabay, 2006). In the bone marrow, IL-6 enhances the production of platelets and the activation of hematopoietic stem cells. Moreover, IL-6 acts on synovial fibroblasts to increase osteoclast differentiation and angiogenesis. IL-6 also stimulates the collagen production by dermal fibroblasts (Tanaka et al., 2012).

However, IL-6 has an important role in the regenerative response of intestinal epithelial cells and hepatocytes to injury (Scheller et al., 2011; Schmidt-Arras and Rose-John, 2016). In addition to its effect on hepatocyte regeneration, IL-6 acts on liver metabolic functions. This cytokine is therefore crucial in the liver homeostasis (Hassan et al., 2014; Schmidt-Arras and Rose-John, 2016). These anti-inflammatory effects of IL-6 can have important consequences on the use of IL-6 inhibitors for the treatment of chronic inflammatory diseases. Indeed, blockade of IL-6 is associated with gastrointestinal perforations (Calabrese and Rose-John, 2014; Taniguchi et al., 2015), transaminase elevation (Genovese et al., 2017) and adverse

31 1.3.2 Tumor necrosis factor αα

TNFα is a pleiotropic cytokine that participates in a variety of inflammatory, infectious and malignant conditions. Activated monocytes and macrophages are the main sources of TNFα but a wide range of cells can also produce TNFα including mast cells, T cells, natural killer (NK) cells and non-immune cells such as endothelial cells (Sedger and McDermott, 2014).

TNFα is synthesized as a 26 kDa membrane bound protein which can be cleaved into a soluble 17 kDa form by the TNFα-converting enzyme (TACE also known as ADAM17).

Both membrane-associated and soluble TNFα are active and mediate their downstream signal by binding to TNFα receptor 1 (TNFR1) and TNFR2 (Bradley, 2008). TNFR1 (also known as CD120a) is expressed ubiquitously and activated by both transmembrane and soluble TNFα.

However, TNFR2 (also known as CD120b) is restricted to specific cell types including immune cells or endothelial cells and binds preferentially the transmembrane form of TNFα in the context of cell-cell interactions (Figure 3). These two receptors contain distinct intracellular domains. TNFR2 lacks a death domain and thus is unable to induce programmed cell death directly whereas TNFR1 signals by the recruitment of TNFR-associated-death domain protein (TRADD) (Kalliolias and Ivashkiv, 2016).

Trimeric TNFα binding to TNFRs leads to receptor trimerization and assembly of distinct signaling complexes: complexes I, IIa, IIb and IIc (Figure 3). The formation of the complex I induces MAPK signaling cascades and the activation of nuclear factor κB (NFκB) pathway leading to expression of genes involved in inflammation, host defense and cell proliferation and survival. In contrast, activation of complexes IIa and IIb (also known as ripoptosome) by the TNFα-TNFR1 binding mediates cell apoptosis through the activation of a caspase cascade. Furthermore, assembly of complex IIc (necrosome) activates the necroptosis effector mixed lineage kinase domain-like protein (MLKL) that results in necroptosis and inflammation (Figure 3). Indeed, necroptosis is characterized by cell membrane rupture leading to the release of intracellular contents triggering local inflammation, in contrast to apoptosis where cells are rapidly phagocytized (Kalliolias and Ivashkiv, 2016).

FIGURE 3: TNFα signaling pathways via TNFα receptor 1 and 2

(a) Both soluble and transmembrane TNFα can activate TNFα receptor (TNFR) 1 signaling. TNFR1 bears a death domain recruiting the TNFR-associated-death domain protein (TRADD). Ligand binding to TNFR1 leads to complex I assembly, which induces mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) driving to inflammation, tissue degeneration, host defense and cell proliferation and survival. In contrast, other signaling pathways are associated with programmed cell death: assembly of Complex IIa and IIb induces apoptosis whereas activation of complex IIc results in necroptosis and inflammation. (b) TNFR2 is preferentially activated by transmembrane TNFα. Ligation of TNFR2 leads to the recruitment of TNFR-associated factor 2 (TRAF2), which triggers assembly of complex I, and activation of MAPK, NFκB and AKT pathways. This mediates homeostasis effects including tissue regeneration, cell proliferation and survival as well as inflammatory and host defense effects. MLKL, mixed lineage kinase domain-like protein. Adapted from Kalliolas and Ivashkiv, 2016 - DOI: 10.1038/nrrheum.2015.169, with permission from RightsLink / Springer Nature; License Number: 4401830967157

TNFα can therefore trigger multiple signaling pathways involved in inflammation, host defense, proliferation and cell death. One of the major biological functions of TNFα is in the immune response to bacterial, viral and parasitic infections. TNFα is a key regulator of the local inflammatory immune response by initiating the release of a cascade of inflammatory mediators, promoting thrombosis and increasing vascular permeability, which enhances immune cell recruitment in the site of infection (Bradley, 2008). TNFα is an attractive and current therapeutic target for a wide range of inflammatory diseases including rheumatoid

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The pro-inflammatory cytokine IL-17 is presented in the part 2 of the introduction.